Voltage-dependent Ca2+ channels (VDCCs) are heteromultimeric complexes present in both neuronal and non-neuronal tissues, including heart and skeletal muscle. VDCCs are minimally composed of three subunits: a pore-forming transmembrane α1 subunit, a hydrophilic intracellular β subunit, and a membrane-associated α2δ subunit; a transmembrane γ subunit is also found in skeletal muscle tissue. Multiple subtypes and/or splice variants of the α1, β, and α2δ subunits have been found.
Gabapentin ((1-aminomethyl)cyclohexane acetic acid or Neurontin) is a structural analogue of GABA, which is mainly used as an adjunctive therapy for epilepsy. Recent research suggests that gabapentin may also have clinical utility for various indications including anxiety and pain. Although designed as a lipophilic GABA-mimetic, gabapentin does not have a high affinity for either GABAA or GABAB receptors, GABA uptake sites, or the GABA-degrading enzyme GABA-transaminase (EC 2.6.1.19).
A novel high affinity binding site for [3H]gabapentin in rat, mouse, and porcin brains has been characterized. Recently, the [3H]gabapentin-binding protein was isolated from pig brain and identified as the α2δ-1 subunit of VDCCs. None of the prototypic anticonvulsant drugs displace [3H]gabapentin binding from the α2δ-1 subunit. [3H]Gabapentin-binding is stereospecifically inhibited by two enantiomers of 3-isobutyl GABA. The rank order of potency of gabapentin, and S- and R-isobutyl GABA, at the [3H]gabapentin binding site mirrors their anticonvulsant activity in mice. However, electrophysiological studies have yielded conflicting data on the action of gabapentin at VDCCs.
The α2δ subunit is derived from a single gene, the product of which is extensively post-translationally modified particularly through the cleavage of the signal sequence. The polypeptide is cleaved to form disulfide-bridged α2 and δ peptides, both of which are heavily glycosylated. Although it seems clear today that the α2 and δ peptides are membrane-associated peptides, it is unclear whether these peptides comprise one or several transmembrane domains. Furthermore, the location, size and structural configuration of these eventual transmembrane domains remains to be determined.
But in any event, the fact that α2δ is a membrane-associated protein, regardless of its precise structural configuration, renders its large scale expression in recombinant systems difficult. Indeed, as the α2δ protein is targeted to the membrane, it requires detergent solubilisation to release it for purification. This important drawback imposes considerable restrictions for any potential applications requiring large amounts of recombinant protein. Furthermore, the various subtypes of α2δ subunits are different proteins with very low homologies. It is therefore extremely difficult to predict their respective behaviors, for example in gene truncation experiments.
The only assay currently available for the screening of ligands that bind the α2δ subunit involves the use of pig membrane extracts as a source of the α2δ subunit. Such an assay presents major inconvenience. Firstly, because the assay material is a membrane extract, it is very difficult to accurately determine the protein composition from one assay preparation to another particularly with regard to the subtype. Also, the presence of various impurities in the assay preparation is a problem in small plate assays. Furthermore, as the protein preparation lacks homogeneity, the interaction between the targeted protein and the assay plate is often quite uneven. This renders the streamlining of the assay in a high throughput format almost impossible to achieve.